(1) Preparation of Cardiomyocytes Using Pluripotent Stem Cells
In general, cardiomyocytes undergo active cell division with beating autonomously before birth, but immediately after birth they lose the ability to divide, and since they have very few undifferentiated stem cells and precursor cells whose growth and differentiation abilities are extremely low, when cardiomyocytes die due to exposure to various forms of stress including myocardial infarction, myocarditis and the like, the lost cardiomyocytes cannot be regenerated. As a result, the surviving cardiomyocytes try to maintain myocardial function through compensatory hypertrophy and the like, but if the stress continues and exceeds an allowable threshold, it leads to further exhaustion and death of cardiomyocytes and a consequent lowering of myocardial function (that is, heart failure).
Heart failure and other types of heart disease are the second leading cause of death in Japan, and prognoses are very poor, with a 5-year survival rate of only about 50% for patients with heart diseases. Therefore, it is hoped that development of highly effective therapies for heart failure will lead to great advances in medical welfare as well as improved medical economics. Conventional therapeutic drugs for heart failure include digitalis preparations that increase the contractive force of the myocardium and xanthine preparations and other heart stimulants, but long-term administration of these drugs is known to make the condition worse because there is too much expenditure of myocardial energy. More recently, mainstream therapy has shifted to β-blockers and ACE inhibitors, which reduce the excess burden on the heart due to stimulation of the sympathetic nervous system and renin-angiotensin system, but these methods only deal with the immediate symptoms and cannot restore damaged cardiac tissue. By contrast, heart transplantation is a fundamental treatment for severe heart failure, but it is one that is difficult to apply commonly because of such problems as the shortage of heart donors, ethical concerns, the physical and financial burden on patients and the like.
Therefore, it would seem that methods of transplantation to replace weakened or lost cardiomyocytes would be extremely useful for the treatment of heart failure. In fact, it is known from animal experiments that when immature cardiomyocytes obtained from fetuses are transplanted into adult cardiac tissue, the transplanted cells function effectively (See Non-Patent Document 1). However, it is difficult to obtain sufficient cardiomyocytes for this method, and application to clinical medicine is also difficult from an ethical standpoint.
Attention has therefore focused in recent years on inducing differentiation of stem cells into cardiomyocytes and using these cells for transplantation. At present it has not yet been possible to clearly identify a population of precursor cells or stem cells capable of producing cardiomyocytes in adult cardiac tissue, so pluripotent stem cells, which are less differentiated and can differentiate into a variety of cells, are considered to be useful for the above method.
Pluripotent stem cells are defined as cells which are capable of indefinite or long-term cell proliferation while remaining in an undifferentiated state in an in vitro culture, which retain normal karyotypes, and which have the ability to differentiate into all of three germ layers (ectoderm, mesoderm and endoderm) under appropriate conditions. The three well-known pluripotent stem cells are embryonic stem cells (ES cells) derived from early-stage embryos, embryonic germ cells (EG cells) derived from primordial germ cells at the embryonic stage, and germline stem cells (GS cells) derived from testes immediately after birth.
In particular, it has long been known that ES cells can be induced to differentiate into cardiomyocytes in vitro. Mouse ES cells were used in most of the early studies. When ES cells are cultured in suspension culture as single cells (individual cells dispersed with no adhesion between cells due to enzyme treatment or the like) without the presence of a differentiation-inhibiting factor such as leukemia inhibitory factor (LIF) or the like, the ES cells adhere to one another and aggregate, forming a structure called embryoid bodies (EBs) which are similar to the early embryonal structures. It is also known that cardiomyocytes with spontaneous beating ability appear when these EBs are cultured in suspension or in adhesion on the surface of culture devices.
ES cell-derived cardiomyocytes prepared as described above exhibit very similar properties to those of immature cardiomyocytes in fetal hearts (See Non-Patent Documents 2 and 3). Moreover, it has been confirmed from animal experiments that when ES cell-derived cardiomyocytes are actually transplanted into adult cardiac tissues, they are highly effective, with results similar to those obtained by transplantation of fetal myocardium (See Patent Document 1; Non-Patent Document 4).
In 1995, Thomson et al. first established ES cells from primates (See Patent Document 2; Non-Patent Document 5), and thus the regeneration therapy using pluripotent stem cell-derived cardiomyocytes has become realistic. Subsequently they also succeeded in isolating and establishing human ES cell lines from early human embryos (See Non-Patent Document 6). Moreover, Gearhart et al. established human EG cell lines from human primordial germ cells (See Non-Patent Document 7; Patent Document 3).
Kehat et al. (See Non-Patent Document 8) and Xu et al. (See Patent Document 4; Non-Patent Document 9) have reported that human ES cells can differentiate into cardiomyocytes in vitro, as mouse ES cells can do. According to these reports, cardiomyocytes derived from human ES cells not only have the ability to beat spontaneously but also express and produce myocardial-specific proteins such as myosin heavy and light chains, α-actinin, troponin I and atrial natriuretic peptide (ANP) and myocardial-specific transcription factors such as GATA-4, Nkx2.5, MEF-2c and the like, and from microanatomical observation and electrophysiological analysis it appears that they retain the properties of immature cardiomyocytes at the fetal stage, and could be used for regenerative therapy.
However, one serious problem remains to be elucidated to use pluripotent stem cell-derived cardiomyocytes for cell transplantation therapy and other purposes. When EBs are formed from ES cells or EG cells by conventional methods, not only cardiomyocytes, but also other types of differentiated cells, such as blood cells, vascular cells, neural cells, intestinal cells, bone and cartilage cells and the like, are developed. Moreover, the proportion of cardiomyocytes in these differentiated cell population is not so high, only about 5% to 20% of the total.
Methods of isolating only cardiomyocytes from a mixture of various kinds of cells include a method of adding an artificial modification to the ES cell genes, conferring drug resistance or ectopic expression, and collecting cells having the properties of cardiomyocytes or precursor cells thereof. For example, by introducing a gene cassette capable of expressing a neomycin (G418) resistance gene under the control of the α-myosin heavy chain promoter into mouse ES cells, Field and his co-researchers established a system in which those ES cells could only survive in medium to which G418 had been added when they differentiated into cardiomyocytes and expressed the α-myosin heavy chain gene (See Patent Document 1; Non-Patent Document 4). 99% or more of G418-resistant cells selected by this method were confirmed to be cardiomyocytes. However, although the purity of the cardiomyocytes is extremely high in this method, the final number of cardiomyocytes obtained is only a few percent of the total cell count, making it difficult to obtain enough amounts of cardiomyocytes for transplantation.
Xu et al. have reported that when human ES cells are treated with 5-azacytidine, the percentage of troponin I-positive cells (candidate cardiomyocytes) in EBs rises from 15% to 44% (See Non-Patent Document 9), but even in this method the percentage of cardiomyocytes in EBs does not exceed 50%. Moreover, 5-azacytidine is a demethylation agent that alters the expression of genes by removing methyl groups bound to DNA, and because it acts directly on the chromosomes, it is not a suitable drug for preparing cells for cell transplantation.
Other methods for producing cardiomyocytes more efficiently from ES cells include, in the case of mouse ES cells, addition of retinoic acid (See Non-Patent Document 10), ascorbic acid (See Non-Patent Document 11), TGFβ, BMP-2 (See Non-Patent Document 12), PDGF (See Non-Patent Document 13) and Dynorphin B (See Non-Patent Document 14) and treatment to increase reactive oxygen species (ROS) (See Non-Patent Document 15) and Ca2+ (See Non-Patent Document 16) in the cells, all of which are known to act positively to induce cardiomyocyte differentiation. However, cardiomyocyte-specific or selective differentiation has not been achieved with any of these methods. Recently, the research group including the inventors has shown that when ES cells are transiently treated with a BMP antagonist, differentiation into cardiomyocytes can be induced more efficiently and selectively than in conventional methods (Patent Document 5; Non-Patent Document 17).
(2) Functional Roles of Wnt Proteins During Differentiation and Development of Cardiomyocytes
Wnt proteins, which are secretory proteins, are members of a protein family group whose presence is widely found not only in vertebrate animals, but also in invertebrate animals such as nematodes and insects, and their gene family is known to have many molecular species (Non-Patent Documents 18 and 19). For example, 19 Wnt genes (Wnt-1, 2, 2b/13, 3, 3a, 4, 5a, 5b, 6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11, 16) have been identified in humans and mice so far. Wnt proteins encoded by these Wnt genes have different tissue specificity, but are structurally similar to each other.
When Wnt proteins contribute as ligands to the intracellular signaling systems, they bind to the seven-transmembrane Frizzled (hereinafter abbreviated as Fzd) family of receptors present on the cell membrane. There are several pathways acting downstream of Fzd receptors, and the most major pathway is inhibition of β-catenin phosphorylation mediated by glycogen synthase kinase (GSK)-3β. In the absence of Wnt signals, β-catenin is captured together with GSK-3β by Axin on Adenomatous polyposis coli (APC) protein and is rapidly phosphorylated by GSK-3β. The phosphorylated β-catenin undergoes ubiquitination and proteasome-mediated degradation.
On the other hand, when Wnt proteins bind to Fzd receptors, an intracellular factor Dishevelled is activated to capture GSK-3β, whereby β-catenin is not phosphorylated and remains in free form within the cytoplasm and further migrates into the nucleus. After migrating into the nucleus, β-catenin binds to lymphoid enhancer factor-1/T cell factor (hereinafter abbreviated as LEF-1/TCF) present in the nucleus to form a transcription activator complex, thereby inducing transcription of a target gene. Such a signaling pathway involving accumulation and nuclear migration of β-catenin is called the “classical” Wnt pathway or the canonical Wnt signaling pathway, and a family of molecular species (e.g., Wnt-1, Wnt-3a, Wnt-8a) capable of activating this pathway is referred to as canonical Wnt. It is also known that activation of the canonical Wnt signaling pathway is induced by treatment with various GSK-3β inhibitors.
Wnt ligands are known to activate not only the β-catenin pathway but also other signaling pathways through Fzd receptors. Such signaling pathways include the planar cell polarity (PCP) pathway which activates JNK (Jun N-terminal kinase), a kind of MAP kinase, and the Ca2+ pathway which elevates the intracellular Ca2+ concentration and activates protein kinase C through trimeric G protein activation and the subsequent phospholipase C activation (Non-Patent Documents 19 and 20). These pathways are called “non-classical” Wnt pathways or non-canonical Wnt signaling pathways, in contrast to the canonical Wnt signaling pathway. Wnt-4 and Wnt-11 have been reported to be Wnt family molecules capable of activating such pathways, and these Wnt ligands act to inhibit the canonical Wnt signaling pathway.
It should be noted that some molecular species of Wnt protein have the ability to activate both canonical and non-canonical pathways, depending on the type of target cells and their differentiation stage, as well as differences in Fzd receptors expressed in the target cells. For example, Wnt-5a is known to act as non-canonical Wnt in commonly used assay systems such as secondary axis formation in Xenopus laevis embryos and carcinogenesis of mammary gland epithelial cells, whereas Wnt-5a has also been reported to induce stabilization of β-catenin and its transcription activity in ES cells, i.e., to activate the canonical Wnt signaling pathway in ES cells (Non-Patent Document 21).
Wnt proteins are known to be involved in a wide variety of biological functions during development, growth and differentiation of various cells, tissues and cancers. Cardiomyocytes develop from a part of the lateral plate mesoderm at the early stage of development, and then repeatedly divide and grow to form a heart. The presence or absence of Wnt signals plays an important role in this process, as demonstrated in several cases. By way of example, in the early stage of avian or Xenopus laevis development, ectopic and/or forced expression of the Wnt-3a or Wnt-8a gene which activates the canonical Wnt signaling pathway significantly inhibits heart formation (Non-Patent Documents 22 and 23).
On the other hand, so-called Wnt antagonists (e.g., Frzb, Dkk-1) which bind to Wnt-3a or Wnt-8a to inhibit its signaling promote heart formation, thus suggesting that canonical Wnt signals act to inhibit myocardial development.
On the contrary, activation of non-canonical Wnt signaling pathways which antagonize canonical Wnt signals is known to positively induce development and differentiation of cardiomyocytes. Pandur et al. (Non-Patent Document 24) have shown that Wnt-11 which activates non-canonical pathways without activating the canonical pathway is a factor essential for heart development in Xenopus laevis. Thereafter, the promoting effect of Wnt-11 has also been confirmed in myocardial differentiation-inducing systems for mouse ES cells (Non-Patent Document 25) and human vascular endothelial precursor cells (Non-Patent Document 26). As to activation of non-canonical Wnt signaling pathways, it is also known that cardiomyocytes can be induced to differentiate from cells of tongue tissue (Patent Document 6).
On the other hand, unlike the above cases, it is known that activation of the canonical Wnt signaling pathway acts to promote myocardial differentiation from embryonic carcinoma cells (EC cells). P19CL6 cells, a subline of P19 cells which are a kind of EC cells, have the property of differentiating into cardiomyocytes under stimulation with dimethyl sulfoxide (DMSO). When Wnt-3a or Wnt-8 was added to medium, P19CL6 cells were promoted to differentiate into cardiomyocytes as β-catenin was stabilized (Non-Patent Document 27). In this system, it is also shown that the time period sufficient for Wnt protein addition is 4 days immediately after induction of differentiation (Non-Patent Document 28).
P19 cell lines have characteristics partially similar to those of ES cells in that they can be induced to differentiate into cardiomyocytes and neurons. However, P19 cell lines do not have the ability to differentiate into a variety of cells or the ability to form chimeras, unlike ES cells. Moreover, P19 cell lines greatly differ from ES cells in terms of cell surface markers, expressed genes and so on. Namely, P19 cell lines may be used as a model system for ES cells in certain experiments, but do not always have the same characteristics as ES cells. Thus, it was not possible to predict, based on scientific grounds, whether the findings obtained in this experimental system could be directly extrapolated to myocardial differentiation-inducing systems for ES cells and other pluripotent stem cells.
Recently, in experimental systems using mouse ES cells, Wnt-3a protein, a member of canonical Wnt, has been reported to promote myocardial differentiation from ES cells when added for 3 days after initiation of differentiation induction (Naito A et al., 28th Annual Meeting of the Molecular Biology Society of Japan, 2005 Dec. 7 to 2005 Dec. 10, Hakata, Japan; Non-Patent Document 30). However, similar studies carried out by us have indicated that there is no significant promoting effect on differentiation (Example 2), and other research groups have also reported that treatment of mouse ES cells with Wnt-3a produces no particularly significant effect on induction of myocardial differentiation (Non-Patent Document 25) or produces an inhibitory effect on myocardial differentiation (Non-Patent Document 29). Namely, it is not clear how activated canonical Wnt pathway caused on myocardial differentiation from ES cells or other pluripotent stem cells. Under these circumstances, no optimum culture method has been established for inducing myocardial differentiation.    Patent Document 1: U.S. Pat. No. 6,015,671    Patent Document 2: U.S. Pat. No. 5,843,780    Patent Document 3: U.S. Pat. No. 6,090,622    Patent Document 4: WO03/06950    Patent Document 5: WO2005/033298    Patent Document 6: JP 2005-224155 A    Non-Patent Document 1: Soonpaa M H et al., Science, 264:98, 1994    Non-Patent Document 2: Maltsev V A et al., Mechanism of Development, 44:41, 1993    Non-Patent Document 3: Maltsev V A et al., Circulation Research, 75:233, 1994    Non-Patent Document 4: Klug M G et al., Journal of Clinical Investigation, 98:216, 1996    Non-Patent Document 5: Thomson J A et al., Proceedings of the National Academy of Sciences of the United States of America, 92:7844, 1995    Non-Patent Document 6: Thomson J A et al., Science, 282:1145, 1998    Non-Patent Document 7: Shamblott M J et al., Proceedings of the National Academy of Sciences of the United States of America, 95:13726, 1998    Non-Patent Document 8: Kehat I et al., Journal of Clinical Investigation, 108:407, 2001    Non-Patent Document 9: Xu C et al., Circulation Research, 91:501, 2002    Non-Patent Document 10: Wobus A M et al., Journal of Molecular and cellular Cardiology, 29:1525, 1997    Non-Patent Document 11: Takahashi T et al., Circulation, 107:1912, 2003    Non-Patent Document 12: Behfar A et al., FASEB Journal, 16:1558, 2002    Non-Patent Document 13: Sachinidis et al., Cardiovascular Research, 58:278, 2003    Non-Patent Document 14: Ventura C et al., Circulation Research, 92:623, 2003    Non-Patent Document 15: Sauer H et al., FEBS Letters, 476:218, 2000    Non-Patent Document 16: Li J et al., Journal of Cell Biology, 158:103, 2002    Non-Patent Document 17: Yuasa S et al., Nature Biotechnology, 23:607, 2005    Non-Patent Document 18: Nusse R, Cell Research, 15:28,    Non-Patent Document 19: Widelitz R, Growth Factors, 23:111, 2005    Non-Patent Document 20: Kühl M et al., Trends in Genetics, 16:279, 2000    Non-Patent Document 21: Hao J et al., Developmental Biology, 290:81, 2006    Non-Patent Document 22: Schneider V A & Mercola M, Genes and development, 15:304, 2001    Non-Patent Document 23: Marvin M J et al., Genes and Development, 15:316, 2001    Non-Patent Document 24: Pandur P et al., Nature, 418:636, 2002    Non-Patent Document 25: Terami H et al., Biochemical and Biophysical Research Communication, 325:968, 2004    Non-Patent Document 26: Koyanagi M et al., Journal of Biological Chemistry, 280:16838, 2005    Non-Patent Document 27: Nakamura T et al., Proceedings of the National Academy of Sciences of the United States of America, 100:5834, 2003    Non-Patent Document 28: Naito A T et al., Circulation Research, 97:144, 2005    Non-Patent Document 29: Yamashita J K et al., FASEB Journal, 19:1534, 2002    Non-Patent Document 30: Naito A T et al., Proceedings of the National Academy of Sciences of the United States of America, 103:19812, 2006